Kinetic Analysis and Mechanism of the Hydrolytic Degradation of Squaramides and Squaramic Acids

Article abstract of DOI:10.1021/acs.joc.6b02963

The hydrolytic degradation of squaramides and squaramic acids, the product of partial hydrolysis of squaramides, has been evaluated by UV spectroscopy at 37 °C in the pH range 3-10. Under these conditions, the compounds are kinetically stable over long time periods (>100 days). At pH >10, the hydrolysis of the squaramate anions shows first-order dependence on both squaramate and OH^-. At the same temperature and [OH^-], the hydrolysis of squaramides usually displays biphasic spectral changes (A → B → C kinetic model) with formation of squaramates as detectable reaction intermediates. The measured rates for the first step (k₁ ≈ 10^-4 M^-1 s^-1) are 2-3 orders of magnitude faster than those for the second step (k₂ ≈ 10^-6 M^-1 s^-1). Experiments at different temperatures provide activation parameters with values of ΔH^? ≈ 9-18 kcal mol^-1 and ΔS^? ≈ -5 to -30 cal K^-1 mol^-1. DFT calculations show that the mechanism for the alkaline hydrolysis of squaramic acids is quite similar to that of amides.

Full text of DOI:10.1021/acs.joc.6b02963

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Article
Kinetic Analysis and Mechanism of the Hydrolytic
Degradation of Squaramides and Squaramic Acids
Marta Ximenis, Emilio Bustelo, Andrés G. Algarra, Manel Vega,
Carmen Rotger, Manuel G. Basallote, and Antonio Costa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02963 • Publication Date (Web): 20 Jan 2017
Downloaded from http://pubs.acs.org on January 20, 2017
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Kinetic Analysis and Mechanism of the Hydrolytic
Degradation of Squaramides and Squaramic Acids.
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†
‡
‡
†
†
Marta Ximenis, Emilio Bustelo, Andrés G. Algarra, Manel Vega, Carmen Rotger,
‡
†
Manuel G. Basallote,* and Antonio Costa.*
†
Department of Chemistry, Universitat de les Illes Balears, Palma, 07122, Spain.
‡
Department of Materials Scientist, Metallurgic Engineering and Inorganic Chemistry,
Universidad de Cádiz, Puerto Real, 11510, Cádiz, Spain.
Eꢀmail: antoni.costa@uib.es
Eꢀmail: manuel.basallote@uib.es
Tel.: +34 971 173266. Fax: +34 971 173426.
KEYWORDS. Squaramides • Squaramic acids • Hydrolysis • Kinetics• DFT
calculations
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ABSTRACT. The hydrolytic degradation of squaramides and squaramic acids, the
product of partial hydrolysis of squaramides, has been evaluated by UV spectroscopy at
37°C in the pH range 3ꢀ10. Under these conditions, the compounds are kinetically stable
over long time periods (>100 d). At pH > 10, the hydrolysis of the squaramate anions
–
shows firstꢀorder dependence on both squaramate and OH . At the same temperature
–
and [OH ], the hydrolysis of squaramides usually displays biphasic spectral changes (A
→
B → C kinetic model) with formation of squaramates as detectable reaction
–4
–1 –1
1
intermediates. The measured rates for the first step (k ~ 10 M s ) are two to three
–
6
–1 –1
orders of magnitude faster than the second one (k
2
~ 10 M s ). Experiments at
‡
different temperatures provide activation parameters with values of ꢀH ~ 9 – 18 kcal
ꢀ1
‡
ꢀ1
ꢀ1
mol and ꢀS ~ (ꢀ5) – (ꢀ30) cal K mol . DFT calculations show that the mechanism
for the alkaline hydrolysis of squaramic acids is quite similar to that of amides.
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Introduction
3,4ꢀDiamino derivatives of squaric acid, also known as squaramides, have received
1
increasing attention in recent years. Owing to their small size and relatively easy
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preparation, squaramides have found applications in diverse areas of chemistry such as
2
1d,3,4
5
molecular recognition, supramolecular catalysis
materials science, ion and
6
7
molecular transport, and sensing. In the biological realm, squaramides have been used
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9
for specific cell labelling, to prepare active bioconjugates, or to replace the phosphate
1
0
and amideꢀtype groups in bioisosteric replacements. In medicinal chemistry, cellꢀ
1
1
penetrating squaramides are known to exhibit significant anticancer and antiparasitic
1
2
activity.
There are evident similarities between squaramides and amides or ureas, the nitrogen
2
atoms of a secondary squaramide are coplanar with the cyclobutendione ring. Their sp ꢀ
2
hybridisation is evidenced by a N–C(sp ) bond length of 1.32 Å, which is significantly
3
13
shorter than the single C(sp )–N bond of 1.46 Å (Figure 1A). The measured barrier to
2
–1
rotation around the N–C(sp ) bond is around 63 kJ mol , which give rise to the
14
observation of
Z,
Z
and
Z,
E
conformers. Squaramides are also known to show certain
5
c,15
aromatic character
and enhanced hydrogen bonding capabilities as hydrogen bond
1
6,13
7a,17
donors
and acceptors
all in all resulting in compounds with excellent hydrolytic
stability.
Despite the growing uses of squaramides and their unique properties, it is surprising that
little is known about the physicochemical events underlying the fate and degradation of
1
8
squaramideꢀbased compounds. Knowledge of the hydrolytic stability and resistance to
chemical degradation of squaramides is a relevant issue, with chemical and biomedical
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implications. In particular, a hydrolytic degradation pathway is likely to occur in
bioactive squaramides and conjugates as part of the catabolism of these compounds.
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Figure 1. a) Xꢀray bond lengths (Å) of a squaramide (CCDC 645873) used as model.
b) Perspective sideꢀview showing the planar arrangement of the squaramide units in the
solid state and the two effective headꢀtoꢀtail NH•••O=C hydrogen bonds occurring
between the squaramide units.
In this work, we have examined the hydrolysis of several squaramides under biological
+
ꢀ
conditions (37 ºC, buffered aqueous solutions, 0.15 M ionic strength, [H ] or [OH ] <
–
2
1
0 M). Nevertheless, the reluctance of these compounds to hydrolysis have led us to
+
ꢀ
strengthen the conditions when necessary (0.01 < [H ] or [OH ] < 1 M), while keeping
the temperature at 37 ºC and raising the ionic strength to 1 M (NaCl).
Results and Discussion
The full hydrolysis of squaramides produces squaric acid and two amines. Nevertheless,
3ꢀhydroxyꢀ4ꢀamino derivatives of squaric acid, also known as squaramic acids, are
formed as intermediates during hydrolysis. Hence, the kinetic data from the hydrolysis
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of squaramic acids are expected to provide valuable information about the rate law and
hydrolysis rates in a simple manner. Furthermore, this allows us to assess the effect of
the neighbouring group, i.e. hydroxyl vs. amide groups. For this reason, the kinetics of
hydrolysis of several squaramic acids as a function of pH has been evaluated before
facing the study of squaramides. In all cases, the kinetics were studied under pseudo
first order conditions by monitoring the timeꢀdependent changes in the UVꢀvis spectra
of the corresponding squaramic acid or squaramide at 37°C. The experiments were
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carried out at different pH values, and the apparent rate constants (
kobs) were calculated
by performing global analyses of the absorption spectra acquired during the
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hydrolysis.
Scheme 1. Alkaline hydrolysis of squaramic acids.
At neutral pH, the squaramic acids
1 – 6 (Scheme 1) show intense absorption bands (ε >
4
ꢀ1
ꢀ1
2
× 10 M cm ) in the UV range with maximum absorptions at 276.0, 283.0, 274.0,
290.7, 294.0 and 308.0 nm respectively. Under the same experimental conditions, the
spectrum of the hydrolysis product, squaric acid, shows a band at
Preliminary kinetic experiments showed that spectral changes are negligible when
samples of squaramic acids to are incubated at 37 ºC for several days in buffered
λmax 269.0 nm.
1
4
solutions ranging from pH 4 – 10. Longꢀterm experiments (40, 100, 190 days) carried
out in screwꢀcapped vials in the same experimental conditions did not evolve into the
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squaric acid either. Hence, it can be deduced that squaramic acids are kinetically stable
towards hydrolysis in mild acidic or basic media. However, hydrolysis of squaramic
ꢀ
acids 1 – 4 occurs at 37 ºC within a few days under stronger alkaline conditions ([OH ]
=
0.01 – 1.00 M). The results clearly reveal the formation of the squarate anion in a
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single kinetic step as depicted in Figure 2a for compound
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The observed rate constants (kobs) show a linear dependence on the concentration of
base (Figure 2b), thus excluding the ionisation of the squaramidic NH hydrogen as it
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would lead to a nonlinear dependence of
fit of the data to Equation 1 leads to the second order rate constants (
Table 1. This shows that the reactivity of the squaramic acids is very similar (5.9 –
.7 × 10 M s ) regardless of the leaving group. However, compound (R = Ph)
exhibits saturation kinetics (equation 2) on hydroxide concentration (Figure 2c), thus
k
obs on the hydroxide ion concentration. The
k) included in
1
– 4
ꢀ6
ꢀ1 ꢀ1
8
5
1
implying the existence of an initial acidꢀbase equilibrium, with equilibrium constant
K,
–
in which OH abstracts a proton from the NH group and form the conjugate base of
5
which is unreactive against hydrolysis. Formation of the squarate anion occurs trough
reaction of the unmodified
defined in equation1.
5
with a rate constant
k
(equation 2), equivalent to the k
-
kobs =k[OH ]
(1)
(2)
-
k[OH ]
kobs
=
-
1
+K[OH ]
Equilibrium and rate constants can be obtained from the fitting to Equation 2, and these
ꢀ6
ꢀ1 ꢀ1
show a faster hydrolysis rate (k = 63 ± 8 ×10 M s ) and an equilibrium constant of 9
ꢀ1
± 1 M .
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ꢀ
5
Figure 2. (a) Spectral evolution for the hydrolysis of a 10 M solution of
4 with 0.9 M
NaOH (for clarity only one out of three recorded spectra is shown). (b) kobs vs. [OH]
plots for
4
1 ꢀ 4 / NaOH, 4 / KOH and 4 / NMe OH. (c) kobs vs. [OH] plot for 5 / NaOH.
A behaviour similar to that of compound
5
has been reported for the alkaline hydrolysis
CONHPh), where K
of trichloroacetamide (CCl CONH ) and trifluoroacetanilide (CF
3
2
3
corresponds to the equilibrium constant for the ionisation process caused by the
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enhanced acidity of these activated amides. Thus, the observed rate equation for
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can
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0, 22
be explained by the formation of its conjugate base as an unreactive side product.
Finally, compound
conditions.
6 does not appreciably hydrolyse under similar experimental
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Table 1. Second order rate constant (k) for the alkaline hydrolysis of 1 – 6 at 37 ºC.
6
1 ꢀ1
Substrate/Base
10 x k2 M s
(
(
(
(
1
2
3
4
)/NaOH
)/NaOH
)/NaOH
)/NaOH
5.9
8.7
6.3
6.7
6.6
7.3
±
±
±
±
±
±
0.1
0.2
0.1
0.2
0.1
0.2
(4)/KOH
(
4
)/NMe
4
OH
a
(
5
)/NaOH
63 ± 8
ꢀ
(
6)/NaOH
a
ꢀ1
A preꢀequilibrium constant of
K = 9 ± 1 M was measured for this system.
The rate constants for the hydrolysis of compounds
1 – 5 compare well with those
reported for standard amides such as acetamide, Nꢀmethylacetamide, and Nꢀ
ꢀ5
ꢀ6
ethylacetamide, which have alkaline hydrolysis constants of 4.71 × 10 , 5.46 × 10 and
ꢀ6
ꢀ1 ꢀ1
23
3
.10 × 10 M s , respectively, at 25ºC. Thus, the anionic nature of the starting
squaramate anion apparently does not interfere the hydrolysis of the squaramide bond.
Additionally, compound
noticeable cation effect.
4
4 4
was treated with KOH/KCl and NMe Cl/NMe OH, without a
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Chart 1. Chemical structures of the squaramides investigated in this work.
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As squaramides derived from simple alkyl or aryl amines are insoluble in water, carbon
chains containing solubilising groups such as dimethylamino or carboxylic acid groups
were introduced to fulfil this function (Chart 1). The spectra of the squaramides
show the absorption band slightly shifted towards higher wavelengths compared to
squaramic acids , the maximum being now observed in the range of 293 – 313 nm.
7 – 14
1
– 6
Longꢀterm experiments were again necessary to follow progress in the hydrolysis of the
squaramides at 37 ºC under buffered neutral (pH 4 to 10) and acidic conditions ([HCl] =
0.01 – 0.14 M). Experiments using samples of compound 10 at different pH in sealed
vials at 37 ºC and measured after 40, 100 and 190 days only showed small changes at
pH 9ꢀ10, and even minor changes at pH lower than 2. Therefore, the hydrolysis of bisꢀ
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squaramides requires stronger conditions under acidic (pH < 2) than under alkaline
conditions (pH > 8).
The alkaline hydrolysis was then studied under conditions similar to those previously
used for squaramates. As expected, the spectral changes observed are more complex
than those found previously for the squaramates. Those for the hydrolysis of the
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2b
antichagasic agent 10
of the spectra with time corresponds to biphasic kinetics. The spectral changes are
satisfactorily fitted to an A C kinetic model with observed rate constants k1obs and
,
are included in Figure 3. For compounds 7 – 11, the evolution
→
B
→
k
2obs
.
Figure 3. a) Spectral changes with time observed during the hydrolysis of squaramide
at 37ºC in NaOH 0.9 M. For clarity, only one out of the three recorded spectra is
shown. b) Selected traces at various wavelengths.
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The rates of both steps vary with the concentration of base, but the dependence is
different in both cases. In the first step, saturation kinetics is observed (Figure 4a), and
k
1obs values fit equation 2. The exception is compound 11, lacking any ionisable NH
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ꢀ
group, for which
k
1obs increases linearly with [OH ] and the values can be fitted to
equation 1 (Figure 4b). In the second step,
k
2obs values satisfactorily fit equation 1 and
yield the second order hydrolysis rate constants k
2
included in Table 2.
ꢀ
Figure 4. Plot of [OH ] vs. k1obs for the hydrolysis of 10 (a) and 11 (b) in the presence of
different salt/base combinations.
The values of k1
, K and k2 for squaramides 7 – 14 are included in Table 2. Remarkably,
the alkaline hydrolysis of trichloroacetamides, acetamides, and Nꢀphenyl ureas, among
21a,23,24
ꢀ
others,
show a nonlinear relationship between k1obs and [OH ] similar to that
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observed for
5
. The saturation kinetics observed in those cases is explained by the
ionisation of the NH groups of the amides or ureas, leading to unreactive conjugate
bases at high pH. A similar interpretation is possible in the present case, the nature of
the preꢀequilibrium responsible for the saturation kinetics at high hydroxide ion
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concentration for
7 – 10 (and also for squaramate 5) is likely related to the ionisation of
the squaramides bearing NꢀH groups (Scheme 2). The different rateꢀlaw observed for
ꢀ
11
, which lacks NH groups and showed a linear dependence on [OH ], gives further
support to this interpretation.
The rate constants for the second resolved step match well with those obtained for the
ꢀ6
ꢀ1 ꢀ1
squaramate hydrolysis, with values around 6×10 M s . A comparison between the
first and second hydrolysis rate constants shows that the first hydrolysis step is roughly
two to three orders of magnitude faster than the second one (k1 >> k2). This effect is
similar to the known difference in reactivity observed for the alkaline hydrolysis of
24
phosphate triesters compared to anionic phosphate diesters. The relative hydrolysis
rates of certain diamides also show a similar trend. For instance, the alkaline hydrolysis
of cisꢀmaleamide at 65 ºC is greater than that for acetamide by two orders of
25
magnitude with the enhanced reactivity being explained based on the electronꢀ
withdrawing ability of a second amide group. However, the same argument cannot be
employed to explain the differences observed between squaramides and squaramate
anions. Instead, it appears that the different rates of hydrolysis of the twoꢀsquaramide
groups have an electrostatic origin. The squaramate anion formed after the first
hydrolysis has more difficulties to react readily with hydroxide or hydroxideꢀwater
cluster anions because of the electrostatic repulsion between the twoꢀnegatively charged
ions.
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Table 2. Kinetic Parameters for the Alkaline Hydrolysis (NaOH) of Squaramides
7– 14.
a
4
6
Substrate
1
0 ×
k
s
1
K
M
10 ×
k
s
2
1 2
k /k
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
1
ꢀ1
ꢀ1
ꢀ1 ꢀ1
M
M
7
8
9
3.42
±
0.02
3.98
±
0.04
7.04
6.22
8.25
±
±
±
0.05
0.09
0.05
49
42
45
84
±
±
±
±
0.6
4
2.6
3.7
5.4
±
0.2
0.2
0.5
3.2
4.6
2.7
±
0.4
±
±
±
±
0.3
0.4
3
10
11
12
6.4
±
0.2
10
29.7
±
0.3
0.001
6.57
±
0.07
452 ± 9
0.141
0.004
±
b
13
14
15.1 ± 0.2
a
–5
The kinetic measurements were carried out at 37°C on 10 M solutions of the
b
corresponding squaramides in H
2
O (1M NaCl). The value of the rate constant for 13 is
an estimation based on the NMR observations.
The k1
/k2 ratios for compounds 7, 8 and 9 are all close to 50 whereas compound 10
shows ratios between 84 and 114, with the value increasing with the size of the cation
+
+
+
(Na < K < NMe
4
)(Supporting Information). Compound 11 exhibits the greatest effect
+
+
(452 for Na and 612 for NMe
4
). All in all, it seems that the presence of
tetramethylammonium cations accelerates the first hydrolysis step in relation to the
second one.
The introduction of aromatic substituents in compounds 12 and 13 provides an
additional stabilization of charged species by both the cyclobutene and phenyl rings.
The formation of an imidate anion makes compound 12 to evolve much more like a
squaramic acid in terms of hydrolysis rate. Besides, the hydrolysis is directed towards
the aliphatic amine group in spite of its slower hydrolysis, which is then followed by a
fast aniline release in the second hydrolysis step.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2. Two-step alkaline hydrolysis of a representative squaramide.
O
O
O
O
O
N
O
N
-O
-O
O-
O
O
N
H
OH-
OH-
+
1
k
1obs
k2obs
H N
2
-O
N
Me N
Me
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Me
NMe2
HN
Me
4
1
0
NMe2
K
−
δ
O
O
δ−
N
N
Me N
Me
2
An interesting aspect that deserves attention is the regioselectivity of the hydrolysis in
unsymmetrical squaramides. The global analysis of the kinetic data is very useful in this
regard, as it provides a calculated spectrum for the reaction intermediate that can be
compared with the experimental spectra of the two possible squaramates. For instance,
the calculated spectrum of the putative intermediate appearing during the alkaline
hydrolysis of 10 shows a maximum at
λ
max = 285.5 nm and therefore does not coincide
max = 276 nm) or max = 290.7
with any of the two available options, squaramates
1
(λ
4 (λ
nm). This observation indicates that the recorded spectrum must be a combination of the
spectra of these two squaramates (Figure S1). Their relative abundances can actually be
obtained by fitting the calculated spectrum to a linear combination of them, and the
results indicate that the mixture is composed of 31% of
1 and 69% of 4.
NMR experiments performed on the squaramide 10 (0.9 M NaOD/D
2
O, 37 °C, 6 h)
confirmed the formation of a mixture of squaramates as intermediates in the alkaline
hydrolysis of unsymmetrical squaramides. Under these conditions, the concentration of
1
the squaramate intermediates approaches a maximum. The resulting HꢀNMR spectrum
matches well with the superposed spectra of 1, 4 and the corresponding free amines
2
(Figure S2). The integration of the characteristic squaramide NCH signals (δ 3.25 –
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3.50 ppm in alkaline conditions) provides a value of 55:45 for the
1:4 ratio, in
reasonable agreement with the previous kinetic results. Although both approximations
do not agree in which is the dominant intermediate, it is clear that the rate constants for
the hydrolysis of both CꢀN bonds are quite similar and that there is no preferred
pathway for the first hydrolysis step. Regarding the hydrolysis of the squaramates, their
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
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35
36
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39
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41
42
43
44
45
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47
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rates are quite similar and in the second kinetic step, the mixture of
1 and 4 would react
6
6
ꢀ1 ꢀ1
with rate constants of 5.9 × 10 and 6.7 × 10 M s , respectively (see Table 1).
However, because of the similarity between both constants, a single averaged value of
6
ꢀ1 ꢀ1
6
.4 × 10 M s is observed for the second kinetic step in the hydrolysis of 10
In marked contrast to squaramides 11, the hydrolysis of compound 12 takes place
with monophasic kinetics, yielding the squarate dianion without any observable
intermediate (Figure S3). The spectral changes can be fitted to an A B kinetic model,
and the 1obs vs. [OH] plot shows a linear dependence on the hydroxide concentration
(Equation 1) with
O/NaOD confirms the absence of detectable intermediates. These results suggest that
.
7
–
→
k
ꢀ5
ꢀ1 ꢀ1
1
k = (1.41 ± 0.01) × 10 M s . NMR monitoring of the reaction in
D
2
the hydrolysis of 12 takes place first at the aliphatic amine side (slow process) and then
at the aniline group (fast), as indicated in Scheme 3. This hypothesis is further supported
by the fact that the value of the rate constant for
5 in Table 1 is larger than the value of
k
1
for 12 in Table 2. The hydrolysis kinetics of 12 is closer to that of the related
ꢀ5
ꢀ1 ꢀ1
squaramate
4
(aliphatic residue,
k
= 0.67 × 10 M s , Equation 1) than to that of 5
ꢀ5
ꢀ1 ꢀ1
(aromatic residue,
k
= 6.3 × 10 M s , Equation 2).
Compound 13 represents another exception to the general behaviour of squaramides.
i
The introduction of two Pr groups in the ortho positions of the aniline substituent
effectively hinders the hydrolysis at both sides of the molecule, which remains unaltered
under similar conditions and time frame (i.e. [OH] = 0.1 – 0.9 M, 37 ºC and 5.5 days).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
After three weeks, NMR experiments show that only 20% of 13 is partially hydrolysed
at the aliphatic amine side, similarly to compound 12 but much slower. In agreement
with the expectations from the behaviour of other compounds, squaramide 14 undergoes
hydrolysis at only one position to yield squaramate 6, which is reluctant to hydrolysis
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
under identical conditions when studied separately. The process takes places in a single
ꢀ
kinetic step with observed rate constants linearly dependent on the [OH ].
Scheme 3. Alkaline hydrolysis of the phenyl substituted squaramide 12.
Variable temperature kinetic experiments allowed obtaining the activation parameters
‡
‡
(
ꢀH and ꢀS ) and the standard enthalpy and entropy of the preꢀequilibrium using Van’t
Hoff plots. Table S1 summarises the activation parameters for compounds 10, and
. The results (ꢀH ~ 12 – 16 kcal mol , ꢀS ~ (ꢀ19) – (ꢀ31) cal K mol ), are similar
7, 9,
‡
ꢀ1
‡
ꢀ1
ꢀ1
11
‡
ꢀ1
‡
to those reported for a series of diamides (ꢀH ~ 9 – 18 kcal mol , ꢀS ~ (ꢀ5) – (ꢀ30) cal
ꢀ1
ꢀ1
25
K mol ), namely oxalamide, malonamide, succinamide and cis/transꢀmaleamide.
This gives further support to the possibility of squaramides and amides sharing a similar
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hydrolysis mechanism with formation of steadyꢀstate tetrahedral intermediates as INT in
26
Scheme 4.
-
Scheme 4. Structure of the tetrahedral intermediate (INT) formed upon OH
attack.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
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45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
-
2-
2-
+ R R NH
O
O
O
O
O
O
O
O
O
TS1
TS
2
1
2
OH-
+
HO
R1
N
R2
O
N
R1
R2
INT
PROD
DFT studies. While the hydrolysis of amides has been extensively studied from a
27
computational viewpoint, to our knowledge the mechanism of hydrolysis of
5
c,28
squaramides and squaramic acids has never been analysed in depth.
Thus, to gain
insight into the mechanism of the hydrolysis of squaramides we carried out DFT
calculations at the M06ꢀ2X//ccꢀpVTZ/PCM level of theory (for further information see
Supporting Information). We initially assumed that the alkaline hydrolysis of
squaramates takes place via the tetrahedral intermediates II formed when the hydroxide
2
9
anion attack the C4 position of a squaramate anion.
The calculations on compounds and , featuring secondary and tertiary alkyl amine
1
6
groups, respectively, only led to minor structural and energetic differences. These
results were further confirmed by computing the hydrolysis of the model compounds 1t
and 6t, in which the alkyl chains were replaced by methyl groups (Table S2 and Figure
S4). In all these cases, the initial formation of the tetrahedral intermediates (INT) is
ꢀ1
ꢀ
thermoneutral (∆GINT ~ 0 kcal mol ), taking place with barriers of ca. 13 – 15 kcal mol
1
(TS
1
). The C
4
 N bond breaking occurs in a subsequent step that is concerted with the
(1) in Figure
proton transfer from the hydroxyl ligand to the departing amine (see TS
2
ꢀ1
S4). This step is rateꢀdetermining and takes place with barriers of ca. 30 kcal mol .
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
It is worth noting that the structures thus computed for the hydrolysis of 1, 1t, 6 and 6t,
27c
are analogous to those calculated for the hydrolysis of amides. Especially relevant are
those of the rateꢀdetermining second step (TS ), all featuring additional fourꢀmembered
2
rings due to the O–H···N interaction that allows the OꢀtoꢀN proton transfer to be
concerted with the C–N bond cleavage. Computational studies of the alkaline amide
hydrolysis have shown that an ancillary water molecule, hydrogen bonded to the
tetrahedral intermediate, significantly decreases this TS2 barrier. The molecule of water
acts as a bridge that facilitates the step due to the formation of more stable sixꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27
membered ring transition states. Importantly, a similar effect is observed when the
alkaline hydrolysis of 1t and 6t is computed in the presence of an explicit H
molecule. The associated energy values are included in Table 3, whereas Figure 5
shows the relevant structures for the hydrolysis of •H O.
1
,
,
6
2
O
1
2
Table 3. Computed relative Gibbs free energies of the minima and transition states
involved in the hydrolysis of squaramates including an explicit water molecule.
-1 a
Values are given in kcal mol .
Substrate
HBA
ꢀ12.6
ꢀ12.2
ꢀ13.4
ꢀ13.8
ꢀ11.7
ꢀ15.7
ꢀ15.7
TS1
5.5
INT
ꢀ7.2
ꢀ7.3
3.6
TS2
9.8
PROD
ꢀ34.8
ꢀ34.9
ꢀ35.1
ꢀ32.4
ꢀ37.0
ꢀ42.5
ꢀ45.0
1
1
6
6
5
1
·H O
2
t
·H
2
O
4.9
8.8
·H
·H
·H
2
O
11.9
4.6
13.6
9.1
t
2
O
ꢀ5.6
ꢀ7.6
ꢀ20.6
ꢀ21.0
2
O
3.9
5.2
b
0t·H
2
2
OꢀA
5.9
ꢀ7.8
ꢀ8.3
b
10t·H
OꢀB
ꢀ6.8
a
Hydroxide anion and Hꢀbonded [squaramate···OH ] species are the reactants, whereas
2
b
the released amine and [squarate···OH ] species are computed as products. The labels
A and B correspond to the two alternative OH attacks on C3 or C4 carbons of model
2
–
squaramide 10t, respectively.
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The interaction between hydroxide and the hydrated squaramates leads to the formation
ꢀ1
of Hꢀbonded adducts (HBA) that are ca. 12 – 15 kcal mol more stable than the
separated reactants. From these adducts, it is possible to generate the tetrahedral
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
1
1
intermediates INT with TS barriers of ca. 18 kcal mol , except for 6 that shows a
ꢀ1
barrier of 25.3 kcal mol . As expected, the explicit H O does not have an active role in
2
the initial step of the hydrolysis, i.e. it is only Hꢀbonded to the squaramate throughout
the step, and therefore the barriers are close to those in Table S2, again except for
In contrast, the data in Table 3 show that the inclusion of an explicit H O molecule in
the calculations decreases the barrier for the second step of the hydrolysis significantly.
6.
2
ꢀ1
At this level of theory, the hydrolysis (TS2) adopts barriers of 10 – 17 kcal mol (cf. 30
ꢀ1
kcal mol in the absence of the H O molecule). Such stabilisation can be explained
2
based on the combination of that due to the Hꢀbonding interactions, also appearing
during the first step of the process, and the aboveꢀmentioned catalyst effect of H
which serves as a proton bridge and facilitates the concerted OꢀtoꢀN proton transfer. The
computed structure of TS (1·H O in Figure 5 clearly exemplifies this catalytic role by
2
O,
)
2
2
presenting H···O distances between the hydroxyl group and the water molecule of 1.15
and 1.29 Å, indicative of an ongoing proton transfer process. Notably, the marked
decrease in the computed TS
2
barriers makes the formation of the tetrahedral
intermediates (TS
1
) rateꢀdetermining in agreement with the experimental results.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Perspective view of the DFTꢀoptimized structures (M06ꢀ2X//ccꢀpVTZ/PCM)
2 1 2 1 2 2 2
of HBA(1·H O), TS (1·H O), INT (1·H O) and TS (1·H O). Distances are given in Å.
Interestingly, the overall computed barrier for the alkaline hydrolysis of squaramate
6 is
ꢀ1
2
7.0 kcal mol , i.e. significantly larger than those of
1,
1t and 6t, and in agreement with
the experimental observation of hindered hydrolysis of this compound. These
i
thermochemical changes can be understood by the presence of the bulkier N( Pr)
2
i
amine. Specifically, the two Pr chains in TS
2
(6·H
2
O)
(Figure S5) preclude the Hꢀ
bonding interaction between the ancillary water molecule and the O atom at one of the
neighbouring C–O groups (d(H···O)= 2.25 Å).
Squaramate
5
(R
1
= Ph), whose alkaline hydrolysis also differs from that of
1
(R
1
=
nBu), has been modelled both in the absence and presence of an ancillary H
2
O
molecule. The resulting free energies (Tables S2 and Table 3) show similarities with
those of compounds and . However, a crucial difference appears regarding the barrier
1
6
for the second step. In the absence of explicit H O molecules, the thermodynamic
2
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differences between TS (5) and TS (1) follow the general trend, TS (5) is 14.7 kcal
2
2
2
ꢀ1
ꢀ1
mol above the separated reactants whereas the value for TS (1) is 28.6 kcal mol . As a
2
ꢀ1
consequence, the overall TS
Comparison of the structures of TS
trace the origin of the effect to the absence in TS
2
barrier for squaramate
(5) (Figure S6), and TS
(5) of the aboveꢀmentioned OꢀH···N
5
is only 16.1 kcal mol .
2
2
(1) (Figure S4) allows to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
interaction (d(H···N)= 2.83 Å, cf. 1.74 Å in TS (1)) that results in the fourꢀmembered
2
ring required for the OꢀtoꢀN proton transfer to be concerted with the C–N bond
30
31
cleavage. Here, the lower basicity of aniline compared to other departing amines,
make the concerted OꢀtoꢀN proton transfer not required for the C N bond cleavage to
take place. As a consequence, the inclusion of a water molecule to model the alkaline
hydrolysis of
5
does not result in the drastic thermodynamic changes computed for
and 6t. Nonetheless, in line with the relatively fast alkaline hydrolysis of (see Table
2
), the calculated TS barrier for this compound in the presence of one explicit H O
1, 1t,
6
5
1
1
ꢀ1
ꢀ1
molecule is 15.9 kcal mol , i.e. 2ꢀ3 kcal mol lower than those for
1, 1t, 6 and 6t (Table
3).
The hydrolysis of the squaramides was computationally analysed subsequently.
Experimental work on the squaramide 10 has shown that the process involves the
formation of two intermediates (squaramates
1 and 4) with comparable rate constants
and, therefore, they both should be formed with relatively similar barriers. To confirm
this hypothesis, we computed the two possible pathways for the first hydrolysis in
alkaline media of the model compound 10t in the presence of one explicit H O
2
molecule.
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1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
Figure 6. Perspective view of the DFTꢀoptimized structures (M06ꢀ2X//ccꢀpVTZ/PCM)
of HBA(10t·H O)ꢀA, TS1(10t·H O)ꢀA, INT1(10t·H O)ꢀA and TS2(10t·H O)ꢀA. Alkyl
2 2 2 2
groups were replaced by methyl groups. Distances are given in Å.
Similarly to the results for the alkaline hydrolysis of squaramates, the interaction of
10t·H O with hydroxide leads to the formation of an Hꢀbonded adduct HBA 10t·H O)
(
2
2
ꢀ1
that is 15.7 kcal mol more stable than the separated reactants (see Figure 6). The attack
of a hydroxide can take place subsequently either at the C3 or C4 positions of this
squaramide, ultimately leading to the formation of the squaramates 1t and 6t
,
respectively. These two pathways have been labelled as A and B, respectively. The
corresponding thermodynamic data are included in Table 3 whereas Figure 6 shows the
structures computed for pathway A. The data indicate that the free energy profiles for
1
pathways A and B are almost identical. Indeed, the TS barriers are 9.8 and 8.9
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kcal/mol, respectively, whereas those for the second step (TS2) are 12.9 and 12.7
kcal/mol, respectively. These minor differences are surely within the computation
errors, and so, the calculations suggest that the products resulting from both pathways,
i.e. squaramates 1t and 6t, should both be observed as the products of the partial
hydrolysis of 10t. These observations give support to the initial hypothesis of the first
alkaline hydrolysis of 10 not being regioselective, i.e. leading to a mixture of
10
11
12
13
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squaramates
1
and
6. From a structural viewpoint, comparison of the geometries for the
first alkaline hydrolysis of 10t·H
2
O in Figure 6 and that of ·H O in Figure 5 shows
1
2
little geometrical differences associated with the nature of the substituent at the adjacent
carbon atom. The electronic consequences of this structural change are, however,
significant as the TS1 and TS2 barriers in 10t·H O are significantly lower than those for
2
6t·H O, in agreement with the first hydrolysis of squaramides being two to three orders
2
of magnitude faster than the second one.
Conclusions
All in all, the studied squaramides and squaramate anions are kinetically stable at a
temperature of 37 °C and pH range of 2 to 8. At pH above 8 the hydrolysis of these
compounds become observable. The present results demonstrate that the alkaline
hydrolysis of squaramate anions proceeds with second order kinetics, and that the
ꢀ6
ꢀ1 ꢀ1
hydrolysis is relatively slow (k ≈ 10 M s ) and unaffected by the existence of
potentially interacting groups such as dimethylamino or carboxylate groups. On the
other hand, the rate constants for the hydrolysis of the squaramides take place in two
4
ꢀ1 ꢀ1
2
kinetically distinct steps. The first step (
k
1
≈ 10 M s ) is ca. 10 times faster than the
ꢀ6
ꢀ1 ꢀ1
second one (k ≈ 10 M s ), with the latter values being comparable to those obtained
2
for the corresponding squaramates. This general trend is not unexpected since the
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second hydrolysis step of squaramides occurs on negatively charged squaramate anions.
26
On these premises, and given the resemblance with the alkaline hydrolysis of amides a
mechanism involving the formation of tetrahedral intermediates through the
nucleophilic attack of a hydroxide ion to the C3 and C4 carbons of the squaramides can
be proposed. This mechanism is supported by DFT calculations using a continuumꢀ
discrete solvation model. In addition to the kinetic and mechanistic details, the present
results demonstrate the kinetic stability of squaramic acids and squaramides in aqueous
solutions in a broad range of pH and must be useful to anyone planning to use them in
the near future.
0
1
2
3
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5
6
7
8
9
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7
8
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6
7
8
9
0
Experimental Section
The various chemicals were of commercial origin (Aldrich or Scharlau) and used as
1
13
13
received. H, C and 2D NMR spectra (at 300 and 600 Mhz) and C (at 75 and 150
MHz) spectra were recorded on 300 and 600 MHz spectrometers in CDCl or ꢀDMSO
solutions at room temperature. The residual proton signal was used as reference.
Chemical shifts ( ) are given in ppm and coupling constants ( ) in Hz. ESIꢀHRMS mass
3
d
6
δ
J
spectra were recorded on magnetic sector or on Orbitrap mass spectrometers. Elemental
analyses (C, H, N) were conducted by the “Centro de Microanálisis Elemental” of the
“Universidad Complutense de Madrid” (Spain).
Synthesis: The squaramic acids
1
–
4
and
6
were prepared by hydrolysis of the
corresponding ethyl esters. Squaramic acid
5 was prepared according to a reported
32
procedure. The squaramides
7
to 11, were previously synthesized according to a
14 are new compounds.
1
2b
modified procedure. Squaramides 12
–
Typical procedure for the preparation of the squaramic acids 1 – 6
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Diethyl squarate (500 mg, 2.94 mmol) in MeCN (3 mL) and one equivalent of the
corresponding amine were mixed in an ovenꢀdried round bottom flask. The mixture was
stirred under nitrogen at room temperature (1a, 2a) for 10 h or at higher temperatures
for a variable period (see below). The crude mixture was then concentrated under
reduced pressure and purified by column chromatography on silica gel eluting with
CH
ethyl esters 1a
0 h (Milliꢀq, 15 – 20 mL), with or without added acid, recovering the resulting solid
acids 1 – 4 by filtration.
2
Cl
2
/MeOH (95:5 v/v), or CH
2 2
Cl ꢀEtOAc (90/10 v/v) to afford the squaramic acid
–
6a. Next, the esters (500 mg) were digested with water at 90 °C for 8 to
2
,
6
1
4
3
ꢀ(butylamino)ꢀ4ꢀethoxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (1a)
.
White amorphous solid 562
1
mg, yield 97%. mp 49ꢀ50 ºC. H NMR (CDCl
3
)
δ
6.42 (br s, 0.74H), 5.32 (br s, 0.26H),
4
1
1
.76 (q,
J
= 6.9 Hz, 2H), 3.65(br, 0.58H), 3.42(br q,
J
= 6.6 Hz, 1.53H), 1.59 (m, 2H),
13
.44 (t,
J
= 6.9 Hz, 3H), 1.36(m, 2H), 0.92 (t,
J
= 7.2 Hz, 3H). C NMR (CDCl3)
δ
89.9, 182.8, 177.6, 172.6, 69.8, 44.8, 32.8, 19.7, 16.0, 13.8. ESI(+)ꢀHRMS m/z (%)
+
calcd for C10
H15NO
3
Na [M+Na] 220.0941; found 220.0936.
1
4
3ꢀ((3ꢀ(dimethylamino)propyl)amino)ꢀ4ꢀethoxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (2a)
.
Yellow
7.76 (br s,
= 6.9 Hz, 2H), 3.78 (br t, 0.7H), 3.54 (br t, =6 Hz,
= 6.9 Hz, 3H).
1
3
amorphous solid 599 mg, yield 90%. mp 56ꢀ59 ºC. H NMR (CDCl ) δ
0.6H), 7.58 (br s, 0.4H), 4.73 (q,
J
J
1.3H), 2.43 (br t,
J
= 5.7 Hz, 2H), 2.22 (s, 6H), 1.73 (m, 2H), 1.42 (t, J
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2
2
2
2
2
2
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
C NMR (CDCl3)
δ
189.5, 183.2, 177.3, 172.6, 69.6, 58.8, 58.3, 45.4, 44.9, 26.8, 16.9.
+
ESI(+)ꢀHRMS m/z (%) calcd for C H N O [M+H] 227.1390; found 227.1389.
1
1
19
2
3
3
8
4
ꢀ((2ꢀethoxyꢀ3,4ꢀdioxocyclobutꢀ1ꢀenꢀ1ꢀyl)amino)butanoic acid (3a)
.
To a solution of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
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4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
diethyl squarate (500 mg, 2.94 mmol) and 4ꢀaminobutiric acid (313 mg, 2.94 mmol) in
MeCN (10 mL), was added DIPEA (1.05 mL). The mixture was stirred at 50°C for 15
h. The crude was concentrated and diluted with water 12 mL and HCl 3N (3 mL). The
resulting solution was extracted with EtOAc (10 × 10 mL). The combined EtOAc
extracts were washed with brine, and dried over Na
2
SO
4
. The solvent was removed
1
under reduced pressure to give a yellow solid 599 mg, yield 90 %. mp 56ꢀ59 °C. H
NMR (CDCl
3
)
δ
7.76 (br s, 0.6H), 7.58 (br s, 0.4H), 4.73 (q,
J = 6.9 Hz, 2H), 3.78 (br t,
0.7H), 3.54 (br t,
J
= 6 Hz, 1.3H), 2.43 (br t, = 5.7 Hz, 2H), 2.22 (s, 6H), 1.73 (m, 2H),
J
1
3
1
5
2
2
.42 (t,
J
= 6.9 Hz, 3H). C NMR (CDCl )
δ
189.5, 183.2, 177.3, 172.6, 69.6, 58.8,
3
–
8.3, 45.4, 44.9, 26.8, 16.0. ESI(–)ꢀHRMS m/z (%) calcd for C10
H12NO
5
[M–H]
+
26.07210; found 226.0717; ESI(+)ꢀHRMS m/z (%) calcd for C10
H
13NO Na [M+Na]
5
50.06859; found 250.0682.
12b
3ꢀ((3ꢀ(dimethylamino)propyl)(methyl)amino)ꢀ4ꢀethoxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (4a)
.
To a solution of diethyl squarate (500 mg, 2.94 mmol) in MeCN (15 mL) were added
N,N, N'ꢀtrimethylꢀ1,3ꢀpropanediamine 341,6 mg (2.94 mmol). The mixture was stirred
at room temperature for 3 h. The crude was concentrated and the residue purified by
column chromatography (Silica gel, CH
Cl
2 2
/MeOH (80/20 v/v) to give a yellow oil 692
= 7.2 Hz, 1H), 4.75 (q, = 7.2 Hz, 1H),
= 7.2 Hz, 1H), 3.34 (s, 1.5H), 3.15 (s, 1.5H), 2.33 (m,
H), 2.24 (s, 3H), 2.22 (s, 3H), 1.81 (m, 2H), 1.45 (t, = 7.2 Hz, 1.5 H), 1.44 (t, = 7.2
188.3, 188.0, 181.5, 175.6, 175.5,
1
mg, yield 98 %. H NMR (CDCl
3
)
δ
4.76 (q,
J
J
3
.72 (t, J = 7.2 Hz, 1H), 3.44 (t, J
2
J
J
13
3
Hz, 1.5 H) ppm. C NMR (75 MHz, CDCl ) δ
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171.65, 171.3, 68.7, 55.4, 49.7, 49.0, 44.4, 36.0, 35.6, 25.0, 24.8, 15.2 ppm. ESI(+)ꢀ
+
HRMS m/z (%) calcd for C H N O Na [M+H] 241.1552; found 241.1557.
12
21
2
3
39
3
ꢀ(ethoxy)ꢀ4ꢀ(phenylamino) cyclobutꢀ3ꢀeneꢀ1,2ꢀdione (5a)
.
To a solution of diethyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
39
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
squarate (500 mg, 2.94 mmol) in MeCN (15 mL) was added aniline (275 mg, 2.95
mmol). The mixture was stirred at 80°C for 24 h to give a yellowꢀorange solid which
was isolated by filtration. The crude solid was digested at room temperature with
hexaneꢀethanol (92/8 v/v; 20 mL) and hexane (2 × 20 mL) and filtered to give a yellow
1
solid 500 mg, yield 78 %. mp 111ꢀ114 °C. H NMR (CDCl
3
)
δ
8.04 (br s, 1H), 7.32 (m,
13
4H), 7.15 (m, 1H), 4.88 (q,
J
= 7.2 Hz, 2H), 1.50 (t,
J
= 7.2 Hz, 3H). C NMR (CDCl
3
)
δ
189.0, 184.1, 178.3, 168.8, 137.2, 129.7, 125.1, 119.5, 70.6, 16.1.
4
0
3
ꢀ(diisopropylamino)ꢀ4ꢀethoxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (6a). To a solution of diethyl
squarate (500 mg, 2.94 mmol) in MeCN (15 mL) were added N,Nꢀdiisopropylamine
598 mg, 5.90 mmol) and DIPEA (1.05 mL). The mixture was stirred at 50°C for 24 h.
(
The crude was concentrated and the residue purified by column chromatography (Silica
1
gel) to give a yellow solid 630 mg, yield 95 %. mp 77ꢀ79 °C. H NMR (CDCl
3
)
δ
4.82
(q,
J
= 7.2 Hz, 2H), 4.63 (br m, 1H), 3.93 (m, 1H), 1.45 (t,
J
δ
= 7.2 Hz, 3H), 1.29 (d,
J =
1
3
6.6 Hz, 6H) + 1.28 (d,
J
= 6.9 Hz, 6H). C NMR (CDCl )
3
189.1, 182.6, 175.7, 171.3,
3
69.6, 50.0, 48.8, 22.1, 21.9, 16.1. ESI(+)ꢀHRMS m/z (%) calcd for C12H19NO Na
+
[
M+Na] 248.1257; found 248.1252.
32
3
ꢀ(butylamino)ꢀ4ꢀhydroxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (1). Obtained as the free acid from
a mixture of 1a (500 mg), water (30 mL) and HCl (3N, 1 mL). White solid 367 mg,
1
yield 86%. mp 165 °C (dec.). H NMR (DMSOꢀd6
)
δ
8.32 (br t, 1H), 3.38 (q,
J
= 6.6
= 7.2 Hz, 3H). C NMR (DMSOꢀ
85.6, 184.9, 183.7, 174.4, 43.7, 32.9, 19.5, 14.0. ESI(–)ꢀHRMS m/z (%) calcd for
13
Hz, 2H), 1.49 (m, 2H), 1.28 (m, 2H), 0.87 (t,
J
6
d ) δ
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
–
C H NO [M–H] 168.06572; found 168.06527. ESI(+)ꢀHRMS m/z (%) calcd for
8
10
3
+
C H NO Na [M+Na] 192.06314; found 192.06311.
8
11
3
3
ꢀ((3ꢀ(dimethylamino)propyl)amino)ꢀ4ꢀhydroxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (2). Pale ochre
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
amorphous solid 430 mg, yield 98%. mp 204ꢀ207 ºC. H NMR (DMSOꢀd
6
)
δ
7.16 (t,
J
=
6.3 Hz, 1H), 3.42 (q, J = 6 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H), 2.75 (s, 6H), 1.83 (m,
13
2
H). C NMR (D
2
O)
δ
197.2, 191.1, 183.9, 57.6, 45.4, 43.2, 28.5. ESI(+)ꢀHRMS m/z
+
(%) calcd for C H N O Na [M+Na] 221.0897; found 221.0887.
9
14
2
3
4ꢀ((2ꢀethoxyꢀ3,4ꢀdioxocyclobutꢀ1ꢀenꢀ1ꢀyl)amino)butanoic acid (3). Obtained from a
mixture of 1a (500 mg), water (30 mL) and HCl (3N, 3 mL). The crude solution was
extracted with EtOAc (10 × 10 mL). The combined EtOAc extracts were washed with
brine and dried with Na
2
SO
4
. Removal of the solvent afforded
3
as a white solid 122
8.28 (br t, 1H), 3.39 (q,
185.2,
NO
1
mg, yield 28 %. mp 157ꢀ159 ºC (dec.). H NMR (DMSOꢀd )
δ
J
6
13
=
6
6.3 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 1.74 (m, 2H). C NMR (DMSOꢀd ) δ
1
84.5, 183.1, 174,0 173.9, 43.0, 30.6, 25.7. ESI(–)ꢀHRMS m/z (%) calcd for C
8
H
8
5
–
[
M–H] 198.04080; found 198.0401.
3
ꢀ((3ꢀ(dimethylamino)propyl)(methyl)amino)ꢀ4ꢀhydroxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (4).
Obtained following the general procedure. Then, the crude solid was digested with hot
1
ethyl ether (3 × 15 mL). White solid 433 mg, yield 98 %. mp >250°C (dec.). H NMR
(D
2
O)
δ 3.75 (t, J = 6.6 Hz, 2H), 3.29 (s, 3H), 3.23ꢀ3.18 (t, J = 7.7 Hz, 2H), 2.91 (s,
1
3
6
H), 2.13 (m, 2H). C NMR (D
2
O)
δ
196.1, 190.5, 183.0, 57.3, 50.3, 45.4, 38.2, 25.0.
+
ESI(+)ꢀHRMS m/z (%) calcd for C H N O [M+H] 213.1239; found 213.1238. Anal
10 17
2
3
Calcd for C H N O : C, 56.59; H, 7.60; N, 13.20; found: C, 56.10; H, 7.30; N, 13.03.
10
16
2
3
3ꢀ(hydroxy)ꢀ4ꢀ(phenylamino) cyclobutꢀ3ꢀeneꢀ1,2ꢀdione (5). Obtained as described in the
32
literature by MW irradiation of a mixture of squaric acid and aniline.
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3ꢀ(diisopropylamino)ꢀ4ꢀethoxycyclobutꢀ3ꢀeneꢀ1,2ꢀdione (6). Crystalline white solid,
1
quantitative yield. mp >210°C (dec). H NMR (DMSOꢀd )
δ 10.31 (br s, 1H), 4.32 (m,
6
13
2H), 1.25 (d,
J
= 6.9 Hz, 12H). C NMR (D
2
O)
δ
193.7, 189.4, 182.2, 51.8, 23.9; ESIꢀ
+
FTMS m/z (%) calcd for C10
m/z (%) calcd for C10
H
16
O
3
N [M+H] 198.1125; found 198.1125. ESI(–)ꢀHRMS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–
H
14
O
3
N [M–H] 196.0979; found 196.0973. Anal Calcd for
10 3
C H15NO : C, 60.90; H, 7.67; N, 7.10: found C, 60.73; H, 7.43; N, 7.06.
General procedure for the preparation of the squaramic acids 7 – 11
The squaramides
7
– 11 were prepared following a modified procedure described
1
2b
previously by us, from equimolar mixtures of diethyl squarate (500 mg, 2.94 mmol)
and the corresponding R
dissolved in a minimum volume of MeCN or EtOH, and stirred for 3 h at room
temperature. After this period, the R NH amine (2.97 mmol) was added and the
1 2
R NH neat amine or. Alternatively, the above mixture was
3 4
R
resulting mixture heated further under stirring for several hours. After cooling at room
temperature, the crude mixture was purified by column chromatography or by selective
precipitation.
3ꢀ(butylamino)ꢀ4ꢀ((3ꢀ(dimethylamino)propyl)(methyl)amino)cyclobutꢀ3ꢀeneꢀ1,2ꢀdione
1
2b
(7).
R
1
R
2
3 4
NH = N,Nꢀdimethylꢀ1,3ꢀpropanediamine (374 µL, 2.94 mmol); R R NH =
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